Regulation of the Min Cell Division Inhibition Complex by the Rcs Phosphorelay in Proteus mirabilis

Bacterial growth conditions.Except where indicated, P. mirabilis and E. coli strains (Table 1) were grown in modified Luria-Bertani (LB) broth (10 g tryptone, 5 g yeast extract, 5 g NaCl per liter) at 37°C with shaking at 250 rpm. All the strains were incubated at 37°C. E. coli strains were grown on 1.5% LB agar, and P. mirabilis strains were grown on 3.0% LB agar to prevent swarming. Antibiotic selection for E. coli contained the following concentrations: 100 μg/ml ampicillin, 20 μg/ml kanamycin, and 25 μg/ml streptomycin. P. mirabilis antibiotic selection used 300 μg/ml ampicillin, 20 μg/ml kanamycin, 35 μg/ml streptomycin, and 15 μg/ml tetracycline. When appropriate, a concentration of 12 μg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) was used for the selection of blue or white colonies for both E. coli and P. mirabilis. For induction of protein expression, a concentration of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was used.

DNA manipulations and transformations.All PCR amplifications were done using Phusion Hot Start II high-fidelity DNA polymerase (Thermo Scientific) and verified by DNA sequencing. All ligations were performed using the Fast-Link DNA ligation kit (Epicentre). For electroporation of plasmids into both E. coli and P. mirabilis, 30 ml of cells was grown to an optical density at 600 nm (OD₆₀₀) of 0.3 to 0.5 in LB broth. The cells were pelleted by centrifugation at 3,000 × g for 5 min at 4°C before being washed twice with 1 ml of ice-cold 10% glycerol and resuspended in 60 μl of ice-cold 10% glycerol per electroporation. Electroporations were done in Bio-Rad Gene Pulser 0.2-cm-diameter cuvettes with a Bio-Rad MicroPulser electroporator. Cells were plated on the appropriate antibiotics after 1.5 h shaking at 250 rpm at 37°C. For P. mirabilis transformations, competent cells were incubated for 30 min with plasmid DNA on ice prior to electroporation. P. mirabilis electroporations were allowed to recover for 3 h.

Construction of strains.For bacterial conjugations, overnight cultures were washed 3 times in 1 ml of fresh LB broth. One hundred microliters of each strain was combined in a 1.5-ml microcentrifuge tube before being added to a predried 1.5% LB agar plate; 200 μl of each strain alone was added to separate plates to serve as controls. After 7 h of incubation at 37°C, the cells were resuspended in 4 ml LB broth. The mating mixture and the individual control strains were plated on 3% LB agar containing the appropriate antibiotics and incubated overnight at 37°C.

To construct a minC mutant, the minC gene was amplified using minC-F and minC-R primers (Table 2) and ligated into pBluescript SK (Stratagene) digested with XbaI and BamHI (New England BioLabs). The cloned minC gene was then mutagenized using an EZ-Tn5 <Kan-2> TNP Transposome kit (Epicentre). A minC::EZ-Tn5 insertion in the center of the minC gene at a position 387 bp into the 702-bp minC open reading frame (ORF) was identified by restriction enzyme analysis and confirmed by DNA sequencing. This is referred to here as minC::Kan^r. This was then subcloned into pKNG101, creating pMinC. pKNG101 is a suicide vector harboring streptomycin resistance, used to select for initial pKNG101 integration, and the sacB gene, which selects for the loss of pKNG101 in the presence of 10% sucrose. pMinC was maintained in E. coli SM10 λpir. This strain was conjugated with wild-type PM7002, and selection for PM7002 harboring the Campbell-type insertion at the minC locus was done on LB plates containing tetracycline (15 μg/ml) and streptomycin (35 μg/ml). An exconjugant was grown in LB broth for only approximately 10 generations and then plated on LSW agar (10 g tryptone, 5 g yeast extract, 5 ml glycerol, and 20 g agar per liter) containing 10% sucrose and kanamycin to select for clones that had lost pMinC but contained the minC locus harboring the EZ-Tn5 Kan^r insertion. Clones that were Str^s Kan^r and sucrose resistant were subjected to Southern blot analysis to confirm they harbored the minC::Kan^r mutation. One of these strains, PMKH1 (PM7002 minC::Kan^r), was used for subsequent experiments.

To construct PMKH4 (PM7002 minC::Kan^r rcsB::Str^r), SM10 pKNG101::rcsB containing an internal fragment of rcsB (10) was conjugated to PMKH1. Exconjugants that were resistant to tetracycline, kanamycin, and streptomycin were confirmed using Southern blot analysis as having a disruption in the rcsB gene.

To complement the minC mutation, the minCDE region was amplified from PM7002 genomic DNA using minCDE-F and minCDE-R (Table 2). The minCDE-containing fragment was cloned into the SmaI site of pBC, creating pBCMinCDE. minCDE was digested out of pBCMinCDE using BamHI and XhoI and cloned into the BamHI/SalI sites of pKNG101, creating pMinCDE (pKNG101 plus minCDE). pMinCDE was maintained in E. coli SM10 λpir and conjugated to PMKH1. Exconjugants that were resistant to tetracycline, kanamycin, and streptomycin were confirmed using Southern blot analysis, and one of the clones, PMKH5, was used for subsequent experiments.

pMinClacZ was created by PCR amplification of the minC upstream region using PminC-F and PminC-R and ligating it into the BamHI and SalI sites of pQF50, a low-copy-number vector harboring a promoterless lacZ that can be used to quantify promoter activities (29). pMinClacZ was electroporated into E. coli DH5α, and clones were selected on LB agar plates containing X-Gal and ampicillin. One of these confirmed clones was purified using the Qiagen Plasmid Mini-Kit, and 5 μl of this DNA was used to electroporate PM7002 and PM7002 rcsB::Str^r, creating PMKH6 and PMKH7, respectively.

5′-RACE.5′ rapid amplification of cDNA ends (5′-RACE) was performed on 1.5 μg of RNA isolated from PM7002 using the 5′-RACE system (Invitrogen) according to version 2.0 of the manufacturer's protocol. First-strand cDNA synthesis was performed using GSP1 (Table 2). PCR amplification of the dCTP-tailed cDNA was done using GSP2 (Table 2) and the provided abridged anchor primer. Nested-PCR amplification of the diluted PCR product was done with Phusion Hot Start II high-fidelity DNA polymerase (Thermo Scientific) using the provided abridged universal amplification primer (AUAP) and GSP2. The resulting product was ligated into the SmaI site of pBC.SK and transformed into E. coli DH5α. The transformant was plated on ampicillin and X-Gal, and white colonies that were confirmed to have the 5′-RACE-generated product were sequenced to identify the 5′ end of the minC gene.

Phase-contrast microscopy.All phase-contrast microscopy images were captured with an Infinity 2-1 charge-coupled-device (CCD) camera (Lumenera) using an Olympus BX51 microscope. For imaging liquid cultures, cells were grown to mid-log phase, and 1.5 μl was pipetted onto a glass slide (VWR International). A coverslip (VWR International) was placed on top of the culture, and images were taken at ×1,000 magnification using oil immersion. Swarm fronts were imaged by adding 650 μl of LB agar to CoverWell imaging chambers (1 by 20 mm by 2.3-mm depth) and then adding 4 μl of an overnight culture to the solidified agar. The cells were grown in a humidified environment at 37°C until they began to swarm. A coverslip was placed on top of the imaging chamber containing the swarming cells, and images were taken at ×1,000 magnification using oil immersion. When applicable, antibiotics and IPTG were added to the media.

β-Galactosidase assays.Overnight cultures were standardized to the same optical density before 300 μl of each culture was spread plated onto 1.5% LB agar containing ampicillin and incubated at 37°C for 2 or 4 h. After the appropriate incubation period, cells were harvested from agar plates using 2 ml of ice-cold LB broth, and 0.9 ml of the harvested culture was pelleted by centrifugation at 13,000 rpm for 4 min. The pelleted cells were placed on ice for 20 min before lysis using chloroform and 0.1% SDS. β-Galactosidase activity was quantified as described by Miller (30).

EMSA.Electrophoretic mobility shift assays (EMSAs) were performed on digoxigenin (DIG)-ddUTP-labeled probes created by PCR amplification using Phusion Hot Start II high-fidelity DNA polymerase (Thermo Scientific) and primers containing EcoRI recognition sites at their ends (Table 2). The amplified DNA was digested with EcoRI for 3 h at 37°C, cleaned up using a PCR purification kit (Qiagen), and boiled for 10 min. After boiling, the digested DNA was placed immediately on ice, and 250 ng was incubated with 1 μl DIG-11-dUTP (Roche), 1 mM dATP (Roche), 4 μl buffer B (6 mM Tris-HCl, 6 mM MgCl₂, 50 mM NaCl, pH 7.5), and 1 μl Klenow enzyme (Roche). The labeling reaction mixture was incubated at 37°C for 1 h, followed by a 10-min incubation at 65°C to heat inactivate the Klenow enzyme. After labeling, the probe was cleaned up using the PCR purification kit.

Binding reactions were done by incubating 0.5 μl of labeled probe with 10× binding buffer (500 mM Tris-HCl, 1 M NaCl, 1 mM EDTA, 0.5 μg/μl bovine serum albumin [BSA], 100 mM dithiothreitol [DTT]), 1 μg/μl poly(dI-dC), and 0 to 320 pmol of purified His-tagged RcsB (RcsB-His₆) brought to a final volume of 20 μl with molecular biology grade water (Fisher). The reaction mixtures were incubated for 30 min at room temperature before being loaded with 5× DNA loading dye on a prerun Mini-Protean 5% Tris-borate-EDTA (TBE) precast gel (Bio-Rad). The gel was run at 100 V for approximately 3 to 4 h at 4°C until the loading dye was no longer visible on the gel. The DNA was transferred onto a 0.22-μm nylon membrane (MagnaGraph) at 15 V with a 0.4-mA limit for 45 min using a Bio-Rad Transblot SD Semi-Dry transfer cell. The DNA was cross-linked to the membrane using a Stratagene cross-linker. Blocking reagent (10×; Roche) was prepared in maleate buffer (100 mM maleic acid [pH 7.5], 150 mM NaCl), and the membrane was blocked for 30 min at room temperature using 1 times blocking solution diluted in maleic acid wash buffer (maleate buffer with 0.3% Tween 20). The membrane was then incubated for 30 min with a 1:1,000 dilution of antidigoxigenin coupled to alkaline phosphatase (Roche) in maleic acid wash buffer. The membrane was washed twice for 15 min each time with maleic acid wash buffer. The membrane was equilibrated in detection buffer (100 mM Tris-HCl [pH 9.5], 100 mM NaCl) for 2 min and then incubated with 1 ml of a 1:10 dilution of CDP-Star (Roche) for 5 min. After briefly blotting the membrane with Whatman paper, it was placed in a sealable plastic bag and exposed to film.

Swarm assays.Overnight cultures were standardized to the same optical density, and 4 μl of each culture was pipetted onto the same predried 1.5% LB agar plate. The culture was allowed to dry into the LB plate before being incubated at 37°C. Measurements of the swarm diameter in centimeters were taken every 30 min until the cells reached consolidation. Antibiotics were included in the agar plate when appropriate. The values represent the average diameter of duplicate sets (4 in total) with standard deviations.

Transcriptome sequencing (RNA-Seq).HI4320 and rcsB::Str^r (PMKM2) cells were harvested at an OD₆₀₀ of 1.1 in triplicate. RNAprotect bacterial reagent was used at a 1:1 ratio (Qiagen). Total RNA was isolated using a MasterPure RNA purification kit (Epicentre), and contaminating DNA was removed using a Turbo DNA-Free kit (Ambion). RNA purity was confirmed on an Agilent 2100 bioanalyzer (Agilent Technologies). DNA-free samples were then enriched for mRNA using a MicrobExpress bacterial mRNA enrichment kit (Ambion). Two rounds of rRNA depletion were performed, and RNA purity was confirmed on an Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA libraries were constructed from HI4320 and rcsB::Str^r mRNA using the SuperScript double-stranded cDNA synthesis kit (Invitrogen). The cDNA libraries were purified by phenol extraction and ethanol precipitation. Sequences were analyzed on an Illumina HiSeq2000 instrument with 50-bp paired-end reads and matched to the HI4320 genome using CLC Genomics Workbench (CLC Bio-Qiagen, Aarhus, Denmark). The total number of reads mapped per transcript was determined and used to determine the reads per kilobase per million reads (RPKM). Three separate sets of RNA samples were analyzed, and the average of samples from two sets was used (data available on request).

qRT-PCR.The same RNA used for RNA-Seq was used for quantitative real-time reverse transcriptase (qRT) PCR. Briefly, the RNA was transcribed into cDNA using an iScript Selected cDNA synthesis kit (Bio-Rad) according to the manufacturer's instructions. qRT-PCR was performed using a 1:10 dilution of the generated cDNA in iQ SYBR green Supermix (Bio-Rad). The reaction was completed in an iCycler iQ Real-Time PCR detection system (Bio-Rad), and the values were normalized to 16S rRNA expression for each sample. The expression of minC, minD, and minE in HI4320 was calculated relative to PMKM2 (HI4320 rcsB::Str^r) using the ΔΔC_T method (31). The primers used to quantify the expression of each gene can be found in Table 2.

Semiquantitative RT-PCR.Total RNA was harvested at hourly intervals (T₁, T₂, T₄, and T₆) after placing the cells on 1.5% LB agar plates using a MasterPure RNA purification kit (Epicentre). The T₀ sample represents cells immediately before being placed on agar surfaces. Contaminating DNA was removed using a Turbo DNA-Free kit (Ambion). The purified RNA was transcribed into cDNA using an iScript Selected cDNA synthesis kit (Bio-Rad). PCR amplification was performed using 16S-F and 16S-R (Table 2), and aliquots from the PCR mixture were taken after 5, 10, and 15 cycles in order to equilibrate cDNA samples relative to 16S rRNA expression. Once equilibrated, minC expression was examined using minC-F and minC-R (Table 2) and flhD expression was examined using flhD-F and flhD-R (Table 2) at cycles 20, 25, 30, and 35.

Western blot analysis.Overnight cultures grown to stationary phase in LB were equilibrated to the same optical density, and then 300 μl was spread onto 1.5% LB agar before incubation at 37°C for 5 h. The cells were gently resuspended in cold LB broth to the same optical density, and 1.5 ml was pelleted and stored at −80°C. The cell pellets were lysed using 400 μl CellLytic B cell lysis reagent (Sigma). Equivalent protein levels for each sample were confirmed using a Bradford assay. The cell lysates were resuspended at a 1:1 ratio in Laemmli sample buffer. Samples were run on a 12% Mini-Protean TGX (Bio-Rad) gel and transferred to a nitrocellulose membrane. The primary antibody used was rabbit anti-FlaA obtained from Robert Belas at the University of Maryland. The secondary antibody was ECL peroxidase-labeled anti-rabbit antibody (Life Sciences).

DAPI (4′,6-diamidino-2-phenylindole) staining and microscopy.Swarmer cells were harvested after 5 h of incubation on 1.5% LB agar and resuspended in 1× phosphate-buffered saline (PBS). The cell suspensions were chemically fixed by the addition of formaldehyde (Sigma) to a 3% (vol/vol) final concentration and incubated for 10 min by gentle rocking. The fixed bacteria were attached to a poly-l-lysine (Sigma-Aldrich)-coated coverglass and incubated with DAPI (AnaSpec, Inc.) at 2 μg/ml (final concentration) in 1× PBS for 5 min in the dark. The excess DAPI solution was removed by washing with 1× PBS. The entire staining procedure was performed at room temperature. The DAPI-stained cells were imaged at room temperature using phase-contrast light and wide-field fluorescence microscopy with an Olympus IX83 microscope. An oil immersion 100× objective and a standard DAPI filter were employed for microscopy. Images were acquired using a Neo 5.5 sCMOS camera (Andor) and MetaMorph software (Molecular Devices). The images were prepared for publication using ImageJ software (NIH).